PROJECT SUMMARY Rapid and reliable identification of pathogenic microorganisms is critical for efficient protection of public health and safety. The high versatility of bacterial pathogens allows them to survive in various environments and the emergence of multidrug-resistant species pose a particularly severe threat. Current identification of bacteria largely relies on phenotypic characterization, Gram staining, culturing, and PCR. However, these techniques are time consuming and require trained laboratory personnel and expensive equipment. Thus, there is a tremendous need to develop a simple and efficient bacterial identification method. Recently, a sensing strategy has emerged that utilizes chemicals that do not specifically interact with a particular analyte, but instead react to the general chemical microenvironment. By using a combination of these chemicals, response patterns can be modelled for highly sensitive and specific detection of chemical and biological analytes. These barcoding arrays or so-called ?chemical noses? often rely upon absorbance or fluorescence intensity as the output, which makes them highly susceptible to the sensor concentration. Innovative methods are required to harness the versatility of chemical barcodes and simultaneously eliminate the pitfalls of concentration dependence. The approach we will employ here is to generate chemical barcodes that come from fluorophores with a ratiometric response, i.e. ratio of fluorescence intensities at different wavelengths that depend on the local chemical environment. Moreover, our recent investigations suggest that dye entrapment in polysaccharide-derived nanoparticles will result in sensors that are stable and able to interact with Gram positive and Gram negative bacteria under various conditions. Thus, the overarching goal of this proposal is to design, synthesize, characterize and evaluate a new sensor array that is based on environment-sensitive ratiometric dyes. The proposed approach will provide a versatile platform for express identification of pathogenic microorganisms in a clinical laboratory setting and in the field. We hypothesize that environment-sensitive fluorescent dyes possessing various substituents will exhibit different spectral responses upon interaction with bacterial cell walls. Being combined into an array, these dyes will produce a unique bar code-like spectral fingerprint for various bacteria, enabling their fast detection and identification. This hypothesis will be tested in three aims that are at the interface of chemistry and microbiology. The first aim is to develop a fluorescent sensor platform that provides a specific multiparametric spectral response with different bacteria. The second aim is to investigate the interactions of the dyes with bacteria to optimize the reproducibility of the sensor response. The third aim is to implement the sensor array as a research tool for probing of bacterial cell envelope homeostasis. Achieving these aims will establish a new class of sensors for rapid and robust bacterial pathogen detection and identification.